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Lactococcus lactis suffers from oxidative stress and riboflavin starvation at elevated temperatures due to dissolved oxygen, which can be relieved partially by exogenously supplied riboflavin. Here we explore whether this phenomenon can be harnessed to obtain riboflavin overproducing mutants. Using a riboflavin auxotrophic L. lactis strain as a riboflavin biosensor, we screened L. lactis cultures that had been exposed to temperature induced oxidative stress for up to one year. Riboflavin secreting mutants could readily be identified, some of which had arisen after just two weeks of exposure to oxidative stress. Whole genome sequencing revealed mutations in the riboswitch, which regulate riboflavin biosynthesis. Riboflavin secretion conferred a significant increase in tolerance to oxidative stress and enabled growth at high temperatures in the presence of dissolved oxygen. It was subsequently demonstrated that vigorous aeration at high temperature (37 °C) could prompt rapid emergence of riboflavin secreting mutants. The protective effect provided by riboflavin against oxidative stress may explain the natural occurrence of lactic acid bacteria (LAB) secreting riboflavin. By optimizing fermentation conditions and eliminating lactate formation, we achieved 64 mg/L riboflavin, the highest reported titer so far for LAB, which indicates great potential for use as a riboflavin fortification agent in food.

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes . Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

Enterococci comprise a group of lactic acid bacteria (LAB) with considerable potential to serve as food fermentation microorganisms. Unfortunately, enterococci have received a lot of negative attention, due to the occurrence of pathogenic and multidrug resistant strains. In this study, we used genomics to select safe candidates among the forty-four studied enterococcal isolates. The genomes of the forty-four strains were fully sequenced and assessed for presence of virulence and antibiotic resistance genes. Nineteen isolates belonging to the species Enterococcus lactis, Enterococcus faecium, Enterococcus durans, and Enterococcus thailandicus, were deemed safe from the genome analysis. The presence of secondary metabolite gene clusters for bacteriocins was assessed, and twelve candidates were found to secrete antimicrobial compounds effective against Listeria monocytogenes isolated from cheese and Staphylococcus aureus. Physiological characterization revealed nineteen industrial potentials; all strains grew well at 42 °C and acidified 1.5 hours faster than their mesophilic counterpart Lactococcus lactis, with which they share metabolism and flavor forming ability. We conclude that a large fraction of the examined enterococci were safe and could serve as excellent food fermentation microorganisms with inherent bioprotective abilities., Competing Interests: Conflicts of interest There are no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis , however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis . NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms., Competing Interests: The authors declare no conflict of interest.

Lactococcus lactis is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, L. lactis can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of L. lactis into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient L. lactis strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and α-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an α-acetolactate decarboxylase deficient L. lactis strain, pyruvate could be converted to α-acetolactate and diacetyl. Summing up, by starving L. lactis for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.

In the manufacture of rennet-coagulated cheese, autolysis is a rate-limiting step for ripening. Previously, a highly autolytic and thermotolerant Lactococcus lactis strain, RD07, was generated, which in preliminary laboratory cheese trials demonstrated great potential as a cheese ripening accelerant. RD07 is proteinase positive (Prt + ) and capable of metabolizing citrate (Cit + ). In this study, we obtained two derivatives of RD07: EC8 lacking the citrate plasmid, and EC2 lacking the proteinase plasmid. EC2 and EC8 retained the autolytic properties of RD07, and autolyzed 20 times faster than Flora Danica (FD) and SD96, where the latter is the parent of RD07. The three strains EC2, EC8 and RD07 were used in a ratio of 90:8:2, to create a simple starter termed ERC. ERC was less sensitive to cooking when cultured in milk and autolyzed well after entering the stationary phase upon facing sugar starvation. The ERC starter was benchmarked against FD and SD96 in laboratory cheese trials. The free amino acid content in cheese prepared using the ERC culture was 31 % and 34 % higher than in cheese prepared using FD and SD96, respectively. Overall, the ERC culture resulted in a more rapid release of free amino acids. A large-scale (5000 L) Gouda cheese trial at a Danish dairy demonstrated that the single strain ERC starter was comparable in performance to FD + an adjunct Lactobacillus helveticus culture. Furthermore, a large-scale Danbo cheese trial demonstrated that ERC could reduce the ripening period by 50 % for long-term ripened (25 weeks) cheese, resulting in better cheese., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anodic electro-fermentation (AEF), where an anode replaces the terminal electron acceptor, shows great promise. Recently a Lactococcus lactis strain blocked in NAD + regeneration was demonstrated to use ferricyanide as an alternative electron acceptor to support fast growth, but the need for high concentrations of this non-regenerated electron acceptor limits practical applications. To address this, growth of this L. lactis strain, and an adaptively evolved (ALE) mutant with enhanced ferricyanide respiration capacity were investigated using an anode as electron acceptor in a bioelectrochemical system (BES) setup. Both strains grew well, however, the ALE mutant significantly faster. The ALE mutant almost exclusively generated 2,3-butanediol, whereas its parent strain mainly produced acetoin. The ALE mutant interacted efficiently with the anode, achieving a record high current density of 0.81 ± 0.05 mA/cm 2 . It is surprising that a Lactic Acid Bacterium, with fermentative metabolism, interacts so well with an anode, which demonstrates the potential of AEF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The presence of lactic acid and galactose makes spray drying of acid whey (AW) a significant challenge for the dairy industry. In this study, a novel approach is explored to remove these compounds, utilizing food-grade microorganisms. For removing lactic acid, Corynebacterium glutamicum was selected, which has an inherent ability to metabolize lactic acid but does so slowly. To accelerate lactic acid metabolism, a mutant strain G6006 was isolated through adaptive laboratory evolution, which metabolized all lactic acid from AW two times faster than its parent strain. To eliminate galactose, a lactose-negative mutant of Lactococcus lactis that cannot produce lactate was generated. This strain was then co-cultured with G6006 to maximize the removal of both lactic acid and galactose. The microbially "filtered" AW could readily be spray dried into a stable lactose powder. This study highlights the potential of utilizing food-grade microorganisms to process AW, which currently constitutes a global challenge., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christian Solem reports financial support was provided by Technical University of Denmark. Christian Solem reports a relationship with Technical University of Denmark that includes: employment., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD + regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

The quality-adjusted time without symptoms or toxicity (Q-TWiST) methodology provides a comprehensive framework for treatment comparison that partitions survival time into distinct health states reflecting both treatment toxicity and disease progression. ZUMA-7 (ClinicalTrials.gov identifier NCT03391466), a phase 3 randomized open-label multicenter study, was conducted to evaluate the efficacy of axicabtagene ciloleucel (axi-cel), a chimeric antigen receptor T cell therapy, compared with standard of care (SOC) involving platinum-based salvage chemotherapy with autologous stem cell transplantation (ASCT) consolidation as a second-line treatment for relapsed/refractory (R/R) large B cell lymphoma (LBCL), and met its primary endpoint of improved event-free survival (EFS). We aimed to use the Q-TWiST method to compare the quality-adjusted survival of R/R LBCL patients treated with axi-cel and those treated with SOC who were enrolled in ZUMA-7. The preplanned analysis of overall survival (OS) was partitioned into 3 mutually exclusive health states: time with grade ≥3 adverse events before the event as defined in the EFS analysis (TOX), time without severe toxicity before the event (TWiST), and time after the event (REL). Q-TWiST was computed as a weighted sum of mean TOX, TWiST, and REL values multiplied by state-specific quality of life (QoL) utility scores. Q-TWiST was evaluated in the intention-to-treat cohort at median follow-up. A relative Q-TWiST gain of 10% was deemed "clinically important" and a gain of ≥15% was deemed "clearly clinically important" based on established categorization. Sensitivity analyses with follow-up ranging from 3 months to the maximum follow-up and subgroup analyses by age and R/R status were explored. At a median follow-up of 23.5 months, the axi-cel cohort showed a significantly longer time without severe toxicity compared with the SOC cohort, with a mean TWiST duration of 11.18 months versus 5.39 months, respectively. The mean TOX was 1.16 months versus .74 months, and mean REL was 6.02 months versus 10.66 months. Quality-adjusted survival was significantly longer with axi-cel by 3.7 months (95% CI, 2.3 to 5.2 months), representing a relative gain of 21.9%. This was reflected across all subgroups, with estimated Q-TWiST gains of 3.1 months (95% CI, 1.5 to 4.9 months) for patients age <65 years, 5.2 months (95% CI, 2.4 to 7.9 months) for those age ≥65 years, 3.2 months (95% CI, 1.4 to 4.9 months) for those with primary refractory disease, 9.1 months (95% CI, 3.9 to months 13.5) for those who relapsed within 6 months, and 4.1 months (95% CI, 1.1 to 7.1 months) for those who relapsed between 6 and 12 months. The Q-TWiST gain for axi-cel also was statistically significant across follow-up durations, increasing from .2 month (95% CI, .1 to .3 month) at a 3-month follow-up to 4.9 months (95% CI, 2.4 to 7.8 months) at the maximum follow-up of 37.7 months. Axi-cel was associated with a statistically significant and "clearly clinically important" gain in quality-adjusted survival, regardless of the relative decline in QoL associated with treatment toxicity, disease progression, or additional cancer treatment. This finding adds to the existing evidence supporting a benefit for axi-cel as a second-line treatment for patients with R/R LBCL., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Plant-based dairy alternatives are gaining increasing interest, e.g. alternatives to yoghurt, cheese, and butter. In all these products butter flavor (diacetyl + acetoin) plays an important role. We previously have reported efficient butter flavor formation from low value dairy side streams using a dairy isolate of Lactococcus lactis deficient in lactate dehydrogenase. Here, we have tested the ability of this strain, RD1M5, to form butter flavor in plant milks based on oat and soy. We found that oat milk, with its high sugar content, supported more efficient production of butter aroma, when compared to soy milk. When supplemented with glucose, efficient butter aroma production was achieved in soy milk as well. We also carried out an extended adaptive laboratory evolution of the dairy strain in oat milk. After two months of adaptation, we obtained a strain with enhanced capacity for producing butter aroma. Despite of its high sugar content, RD1M5 and its adapted version only metabolized approximately 10% of the fermentable sugars available in the oat milk, which we found was due to amino acid starvation and partly starvation for vitamins. The study demonstrates that dairy cultures have great potential for use in plant-based fermentations., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Streptococcus thermophilus is a fast-growing lactic acid bacterium (LAB) used in yoghurt and cheese manufacturing. Recently, we reported how this bacterium could serve as a cell catalyst for hydrolyzing lactose when permeabilized by nisin A. To enhance the lactose hydrolyzing activity of S. thermophilus , we mutated a dairy strain and screened for variants with elevated β-galactosidase activity. Two isolates, ST30-8 and ST95, had 2.4-fold higher activity. Surprisingly, both strains were able to hydrolyze lactose when used as whole-cell lactase catalysts without permeabilization, and ST30-8 hydrolyzed 30 g/L lactose in 6 h at 50 °C using 0.18 g/L cells. Moreover, both strains hydrolyzed lactose while growing in milk. Genome sequencing revealed a mutation in l-lactate dehydrogenase, which we believe hampers growth and increases the capacity of S. thermophilus to hydrolyze lactose. Our findings will allow production of sweet lactose-reduced yoghurt without the use of costly purified lactase enzymes.

Nisin has a tendency to associate with the cell wall of the producing strain, which inhibits growth and lowers the ceiling for nisin production. With the premise that resistance to the cationic chlorhexidine could reduce nisin binding, variants with higher tolerance to this compound were isolated. One of the resistant isolates, AT0606, had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Characterization revealed that AT0606 had an overall less negatively charged and thicker cell wall, and these changes appeared to be linked to a defect high-affinity phosphate uptake system, and a mutation inactivating the oleate hydratase. Subsequently, the potential of using AT0606 for cost efficient production of nisin was explored, and it was possible to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production., (Copyright © 2022. Published by Elsevier Ltd.)

Lactic acid bacteria (LAB) are important members in synthetic microbial consortia due to their 'generally recognized as safe' status and diverse metabolic activities. Defined communities with LAB show great potential in elucidating metabolic interactions that drive their assembly and demonstrating power to address sustainability challenges in food, environment, and health., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lactococcus lactis subsp. lactis (referred to here as L. lactis) is a model lactic acid bacterium and one of the main constituents of the mesophilic cheese starter used for producing soft or semihard cheeses. Most dairy L. lactis strains grow optimally at around 30°C and are not particularly well adapted to the elevated temperatures (37 to 39°C) to which they are often exposed during cheese production. To overcome this challenge, we used adaptive laboratory evolution (ALE) in milk, using a setup where the temperature was gradually increased over time, and isolated two evolved strains (RD01 and RD07) better able to tolerate high growth temperatures. One of these, strain RD07, was isolated after 1.5 years of evolution (400 generations) and efficiently acidified milk at 41°C, which has not been reported for industrial L. lactis strains until now. Moreover, RD07 appeared to autolyze 2 to 3 times faster than its parent strain, which is another highly desired property of dairy lactococci and rarely observed in the L. lactis subspecies used in this study. Model cheese trials indicated that RD07 could potentially accelerate cheese ripening. Transcriptomics analysis revealed the potential underlying causes responsible for the enhanced growth at high temperatures for the mutants. These included downregulation of the pleiotropic transcription factor CodY and overexpression of genes, which most likely lowered the guanidine nucleotide pool. Cheese trials at ARLA Foods using RD01 blended with the commercial Flora Danica starter culture, including a 39.5°C cooking step, revealed better acidification and flavor formation than the pure starter culture. IMPORTANCE In commercial mesophilic starter cultures, L. lactis is generally more thermotolerant than Lactococcus cremoris, whereas L. cremoris is more prone to autolysis, which is the key to flavor and aroma formation. In this study, we found that adaptation to higher thermotolerance can improve autolysis. Using whole-genome sequencing and RNA sequencing, we attempt to determine the underlying reason for the observed behavior. In terms of dairy applications, there are obvious advantages associated with using L. lactis strains with high thermotolerance, as these are less affected by curd cooking, which generally hampers the performance of the mesophilic starter. Cheese ripening, the costliest part of cheese manufacturing, can be reduced using autolytic strains. Thus, the solution presented here could simplify starter cultures, make the cheese manufacturing process more efficient, and enable novel types of harder cheese variants.

The design-build-test-learn (DBTL) cycle has been implemented in metabolic engineering processes for optimizing the production of valuable compounds, including food ingredients. However, the use of recombinant microorganisms for producing food ingredients is associated with different challenges, e.g., in the EU, a content of more than 0.9% of such ingredients requires to be labeled. Therefore, we propose to expand the DBTL cycle and use the "learn" module to guide the development of non-engineered strains for clean label production. Here, we demonstrate how this approach can be used to generate engineered and natural cell factories able to produce the valuable food flavor compound - butanedione (diacetyl). Through comprehensive rerouting of the metabolism of Lactococcus lactis MG1363 and re-installment of the capacity to metabolize lactose and dairy protein, we managed to achieve a high titer of diacetyl (6.7 g/L) in pure dairy waste. Based on learnings from the engineering efforts, we successfully achieved the production of diacetyl without using recombinant DNA technology. We accomplish the latter by process optimization and by relying on high-throughput screening using a microfluidic system. Our results demonstrate the great potential that lies in combining metabolic engineering and natural approaches for achieving efficient production of food ingredients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD , encoding the enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element ( cre ) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positive bacteria, is repressed by phosphorylation by EIIA mtl , and when we knocked out mtlF , we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

Lactose-free dairy products are in great demand worldwide due to the high prevalence of lactose intolerance. To make lactose-free dairy products, commercially available β-galactosidase enzymes, also termed lactases, are used to break down lactose to its constituent monosaccharides, glucose and galactose. In this mini-review, the characteristics of lactase enzymes, their origin, and ways of use are discussed in light of their potential for hydrolyzing lactose. We also discuss whole-cell lactase catalysts, which appear to have great potential in terms of cost reduction and convenience, and which are more natural alternatives to purified enzymes. Lactic acid bacteria (LAB) already used in food fermentations seem to be optimal candidates for whole-cell lactases. However, they have not been industrially exploited yet due to technical hurdles. For whole-cell lactases to be efficient, the lactase enzymes inside the cells must be made available for lactose hydrolysis, and thus, cells need to be permeabilized or disrupted prior to use. Here we review state-of-the-art approaches for disrupting or permeabilizing microorganisms. Lastly, based on recent scientific achievements, we propose a novel, resource-efficient, and low-cost scenario for achieving lactose hydrolysis at a dairy plant using a LAB whole-cell lactase.Key points• Lactases (β-galactosidase) are essential for producing lactose-free dairy products• Novel permeabilization techniques facilitate the use of LAB lactases• Whole-cell lactase catalysts have great potential for reducing costs and resources Graphical abstract.

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn 2+ and 100 mM Ca 2+ , respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

Purpose: This analysis evaluated the psychometric properties of the EQ-5D-3L among patients with Mycobacterium avium complex lung disease (MAC-LD)., Methods: Data from the Phase III CONVERT trial were analyzed. Study measures including the EQ-5D-3L, St. George's Respiratory Questionnaire (SGRQ), the 6-minute walk test (6MWT), and percent predicted forced expiratory volume in 1 second (ppFEV 1 ) were collected at baseline, month 3 (M3), month 4 (M4; only 6MWT), and month 6 (M6). Item characteristics and validity were evaluated at baseline. Test-retest reliability was assessed using intraclass correlation coefficients (ICC) and a weighted kappa statistic among a subgroup of stable patients. Validity was tested by evaluating correlations of the EQ-5D-3L index/visual analogue scale (VAS) scores with SGRQ and 6MWT and comparing mean index/VAS scores across known groups defined using 6MWT and ppFEV 1 . Responsiveness of the EQ-5D-3L was assessed using 6MWT, SGRQ, ppFEV 1 , and culture conversion as anchors., Results: The index score was subject to ceiling effects, with 32.6% of patients reporting perfect health at baseline. ICCs for the index (0.80) and VAS (0.85) scores and weighted kappas for the domains (0.5-0.72) indicated adequate test-retest reliability. Correlations between the index/VAS scores and related domains of the SGRQ and 6MWT were as hypothesized (0.31-0.62), and the mean index/VAS scores were significantly different between the 6MWT and ppFEV 1 known groups (p<0.05), supporting the validity of the EQ-5D-3L. No evidence was found supporting the responsiveness of the EQ-5D-3L to changes in any of the anchors., Conclusion: EQ-5D-3L exhibited evidence of validity and reliability but poor responsiveness to clinically meaningful changes in patients with MAC-LD., Competing Interests: AS, XN, RS, and CS are full-time employees of Pharmerit, the institution which received funding from Insmed Incorporated for conducting the study. PW and MO are full-time employees of Insmed Incorporated. The authors report no other conflicts of interest in this work., (© 2021 Shah et al.)

Microbial production of commodity chemicals has gained increasing attention and most of the focus has been on reducing the production cost. Selecting a suitable microorganism, which can grow rapidly on cheap feedstocks, is of key importance when developing an economically feasible bioprocess. We chose Lactococcus lactis , a well-characterized lactic acid bacterium, as our microbial host to produce pyruvate, which is a commodity chemical with various important applications. Here we report the engineering of Lactococcus lactis into becoming an efficient microbial platform for producing pyruvate. The strain obtained, FS1076 (MG1363 Δ 3 ldh Δ pta Δ adhE Δ als ), was able to produce pyruvate as the sole product. Since all the competitive pathways had been knocked out, we achieved growth-coupled production of pyruvate with high yield. More than 80 percent of the carbon flux was directed toward pyruvate, and a final titer of 54.6 g/L was obtained using a fed-batch fermentation setup. By introducing lactose catabolism into FS1076, we obtained the strain FS1080, which was able to generate pyruvate from lactose. We then demonstrated the potential of FS1080 for valorizing lactose contained in dairy side-streams, by achieving a high titer (40.1 g/L) and high yield (78.6%) of pyruvate using residual whey permeate (RWP) as substrate. The results obtained, show that the L. lactis platform is well-suited for transforming lactose in dairy waste into food-grade pyruvate, and the yields obtained are the highest reported in the literature. These results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) to valuable products., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Suo, Liu, Chen, Li, Solem and Jensen.)

Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain important tasks in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum to produce ethanol, we attained a low yield (63% of the theoretical) when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-product formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with that from Escherichia coli , which is active only under aerobic conditions. However, this tampering resulted in a 10-times-reduced glycolytic flux as well as a greatly increased NADH/NAD + ratio. When we replaced glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly, to 92% of the theoretical. Based on that, we managed to rechannel the metabolism of C. glutamicum into d-lactate with high yield, 98%, which is the highest that has been reported. It is further demonstrated that the PPP-inactivated platform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into a valuable product. IMPORTANCE The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under nongrowing conditions, and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox-balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, d-lactate, and alanine using the dairy waste whey permeate as the feedstock. The production yield was high, and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use nongrowing C. glutamicum cells for producing valuable compounds. Overall, we describe the benefits of disrupting the oxidative PPP in nongrowing C. glutamicum and provide a feasible approach toward waste valorization., (Copyright © 2020 American Society for Microbiology.)

β-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

Lactococcus lactis subsp. lactis biovar diacetylactis is widely used in dairy fermentations as it can form the butter aroma compounds acetoin and diacetyl from citrate in milk. Here, we explore the possibility of producing acetoin from the more abundant lactose. Starting from a dairy isolate of L. lactis biovar diacetylactis, we obtained a series of mutants with low lactate dehydrogenase ( ldh ) activity. One isolate, RD1M5, only had a single insertion mutation in the ldh gene compared to its parental strain as revealed by whole genome resequencing. We tested the ability of RD1M5 to produce acetoin in milk. With aeration, all the lactose could be consumed, and the only product was acetoin. In a simulated cheese fermentation, a 50% increase in acetoin concentration could be achieved. RD1M5 turned out to be an excellent cell factory for acetoin and was able to convert lactose in dairy waste into acetoin with high titer (41 g/L) and high yield (above 90% of the theoretical yield). Summing up, RD1M5 was found to be highly robust and to grow excellently in milk or dairy waste. Being natural in origin opens up for applications within dairies as well as for safe production of food-grade acetoin from low-cost substrates.

Mannitol can be obtained as a by-product of certain heterolactic lactic acid bacteria, when grown on substrates containing fructose. Lactococcus lactis , a homolactic lactic acid bacterium, normally does not form mannitol but can be persuaded into doing so by expressing certain foreign enzyme activities. In this study, we find that L. lactis has an inherent capacity to form mannitol from glucose. By adaptively evolving L. lactis or derivatives blocked in NAD + regenerating pathways, we manage to accelerate growth on mannitol. When cells of the adapted strains are resuspended in buffer containing glucose, 4-58% of the glucose metabolized is converted into mannitol, in contrast to nonadapted strains. The highest conversion was obtained for a strain lacking all major NAD + regenerating pathways. Mannitol had an inhibitory effect on the conversion, which we speculated was due to the mannitol uptake system. After its inactivation, 60% of the glucose was converted into mannitol by cells suspended in glucose buffer. Using a two-stage setup, where biomass first was accumulated by aerated culturing, followed by a nonaerated phase (static conditions), it was possible to obtain 6.1 g/L mannitol, where 60% of the glucose had been converted into mannitol, which is the highest yield reported for L. lactis .

Background: The secondary alcohol 2-butanol has many important applications, e.g., as a solvent. Industrially, it is usually made by sulfuric acid-catalyzed hydration of butenes. Microbial production of 2-butanol has also been attempted, however, with little success as witnessed by the low titers and yields reported. Two important reasons for this, are the growth-hampering effect of 2-butanol on microorganisms, and challenges associated with one of the key enzymes involved in its production, namely diol dehydratase., Results: We attempt to link the metabolism of an engineered Lactococcus lactis strain, which possesses all enzyme activities required for fermentative production of 2-butanol from glucose, except for diol dehydratase, which acts on meso -2,3-butanediol (mBDO), with that of a Lactobacillus brevis strain which expresses a functional dehydratase natively. We demonstrate growth-coupled production of 2-butanol by the engineered L. lactis strain, when co-cultured with L. brevis . After fine-tuning the co-culture setup, a titer of 80 mM (5.9 g/L) 2-butanol, with a high yield of 0.58 mol/mol is achieved., Conclusions: Here, we demonstrate that it is possible to link the metabolism of two bacteria to achieve redox-balanced production of 2-butanol. Using a simple co-cultivation setup, we achieved the highest titer and yield from glucose in a single fermentation step ever reported. The data highlight the potential that lies in harnessing microbial synergies for producing valuable compounds., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The genome of Lactococcus lactis subsp. lactis bv. diacetylactis SD96, a strain used for cheese production, is presented. SD96 is refractory to phage attack, which is a desired property for starter bacteria. Its 10 plasmids provide industrially important traits, such as lactose and citrate metabolism, proteolytic activity, and phage resistance., (Copyright © 2020 Dorau et al.)

Purpose: Advanced non-small-cell lung cancer (aNSCLC; stage IIIB/IV) presents a substantial clinical burden to society; reliable estimates of its economic burden are lacking. Therefore, this study aimed to quantify real-world health care resource utilization (HCRU) and costs of patients with squamous (SQ) and non-SQ (NSQ) aNSCLC who received two or more lines of treatment (2L+) in Europe, and to describe cost-predictors., Methods: The LENS (Leading the Evaluation of Non-squamous and Squamous NSCLC) retrospective chart review study collected data from 2L+ patients with aNSCLC diagnosed between 07/2009 and 08/2011 (wave 1) or 07/2010 and 09/2012 (wave 2) in France, Germany, Italy, Spain, England, the Netherlands, and Sweden. Patients were followed from diagnosis through most recent visit/death. A weighted average of country-specific unit costs (2018 Euro) was applied to systemic anti-cancer therapy usage and HCRU (hospital/emergency department visit, surgery, radiotherapy, ancillary care, biomarker testing) to determine the total cost from aNSCLC diagnosis to death. Generalized linear models (gamma distribution, log link) were used to assess clinical and demographic predictors., Results: Of 973 2L+ aNSCLC patients, median overall survival (OS) was 1.5 years from advanced diagnosis (range: 0.2-5.3; median OS: 1.4 [SQ], 1.6 [NSQ]), 79.0% died during follow-up. Weighted mean total per-patient costs were €21,273, ranging from €17,761 (England) to €30,854 (Sweden), and €15,446 (SQ) to €26,477 (NSQ). Systemic drug costs comprised 77.4% of total costs. Insurance status, presence of epidermal growth factor receptor (EGFR ) mutation, SQ histology, age, alcohol abuse, and year of diagnosis were significant predictors for lower total costs per patient-month, Eastern Cooperative Oncology Group performance status (ECOG PS) ≥1 and country for higher costs., Conclusion: In the era pre-immunotherapy, HCRU and costs were substantial in aNSCLC 2L+ patients, with most of the costs accrued prior to start of 2L. NSQ patients incurred significantly higher total costs than SQ patients in all participating countries., Competing Interests: Katharina Verleger, Caitlyn Solem, and Linlin Luo are employees of Pharmerit, which was contracted by Bristol-Myers Squibb to conduct the retrospective chart review study. Cynthia Macahilig is an employee of Medical Data Analytics, which was contracted by Bristol-Myers Squibb to collect and consolidate the data for this study. Nadine Hertel, Melinda Manley Daumont, and John R Penrod are employees of Bristol-Myers Squibb, a global biopharmaceutical company that researches and develops medicines for NSCLC. The authors report no other conflicts of interest in this work., (© 2020 Verleger et al.)

White biotechnology relies on the sophisticated chemical machinery inside living cells for producing a broad range of useful compounds in a sustainable and environmentally friendly way. However, despite the impressive repertoire of compounds that can be generated using white biotechnology, this approach cannot currently fully replace traditional chemical production, often relying on petroleum as a raw material. One challenge is the limited number of chemical transformations taking place in living organisms. Biocompatible chemistry, that is non-enzymatic chemical reactions taking place under mild conditions compatible with living organisms, could provide a solution. Biocompatible chemistry is not a novel invention, and has since long been used by living organisms. Examples include Fenton chemistry, used by microorganisms for degrading plant materials, and manganese or ketoacids dependent chemistry used for detoxifying reactive oxygen species. However, harnessing biocompatible chemistry for expanding the chemical repertoire of living cells is a relatively novel approach within white biotechnology, and it could potentially be used for producing valuable compounds which living organisms otherwise are not able to generate. In this mini review, we discuss such applications of biocompatible chemistry, and clarify the potential that lies in using biocompatible chemistry in conjunction with metabolically engineered cell factories for cheap substrate utilization, improved cell physiology, efficient pathway construction and novel chemicals production., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl., Results: By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate., Conclusions: An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.

Lactose, the sugar contained in milk, has a low sweetness. We have constructed an efficient whole-cell catalyst (WCC) that can be grown on dairy waste and that is able to convert lactose into a mixture of sugars as sweet as sucrose. The WCC is based on Corynebacterium glutamicum ATCC13032, which has been engineered to metabolize lactose, to express xylose and arabinose isomerase, and to eliminate byproduct formation. When introduced in concentrated cheese whey permeate, its content of 98 g/L lactose was completely hydrolyzed and the liberated sugars partially isomerized into 23.5 g/L fructose and 20.4 g/L tagatose, which corresponds to a 49% conversion of the glucose and a 44% conversion of galactose. The latter is similar to what can be obtained using purified enzymes. The new technology enables better resource utilization and allows for dairy waste to be converted into a valuable food sweetener with many potential uses.

Lactic Acid Bacteria (LAB) are extensively employed in the production of various fermented foods, due to their safe status, ability to affect texture and flavor and finally due to the beneficial effect they have on shelf-life. More recently, LAB have also gained interest as production hosts for various useful compounds, particularly compounds with sensitive applications, such as food ingredients and therapeutics. As for all industrial microorganisms, it is important to have a good understanding of the physiology and metabolism of LAB in order to fully exploit their potential, and for this purpose, many systems biology approaches are available. Systems metabolic engineering, an approach that combines optimization of metabolic enzymes/pathways at the systems level, synthetic biology as well as in silico model simulation, has been used to build microbial cell factories for production of biofuels, food ingredients and biochemicals. When developing LAB for use in foods, genetic engineering is in general not an accepted approach. An alternative is to screen mutant libraries for candidates with desirable traits using high-throughput screening technologies or to use adaptive laboratory evolution to select for mutants with special properties. In both cases, by using omics data and data-driven technologies to scrutinize these, it is possible to find the underlying cause for the desired attributes of such mutants. This review aims to describe how systems biology tools can be used for obtaining both engineered as well as non-engineered LAB with novel and desired properties.

Background: Delactosed whey permeate (DWP) is a side stream of whey processing, which often is discarded as waste, despite of its high residual content of lactose, typically 10-20%. Microbial fermentation is one of the most promising approaches for valorizing nutrient rich industrial waste streams, including those generated by the dairies. Here we present a novel microbial platform specifically designed to generate useful compounds from dairy waste. As a starting point we use Corynebacterium glutamicum, an important workhorse used for production of amino acids and other important compounds, which we have rewired and complemented with genes needed for lactose utilization. To demonstrate the potential of this novel platform we produce ethanol from lactose in DWP., Results: First, we introduced the lacSZ operon from Streptococcus thermophilus, encoding a lactose transporter and a β-galactosidase, and achieved slow growth on lactose. The strain could metabolize the glucose moiety of lactose, and galactose accumulated in the medium. After complementing with the Leloir pathway (galMKTE) from Lactococcus lactis, co-metabolization of galactose and glucose was accomplished. To further improve the growth and increase the sugar utilization rate, the strain underwent adaptive evolution in lactose minimal medium for 100 generations. The outcome was strain JS95 that grew fast in lactose mineral medium. Nevertheless, JS95 still grew poorly in DWP. The growth and final biomass accumulation were greatly stimulated after supplementation with NH 4 + , Mn 2+ , Fe 2+ and trace minerals. In only 24 h of cultivation, a high cell density (OD 600 of 56.8 ± 1.3) was attained. To demonstrate the usefulness of the platform, we introduced a plasmid expressing pyruvate decarboxylase and alcohol dehydrogenase, and managed to channel the metabolic flux towards ethanol. Under oxygen-deprived conditions, non-growing suspended cells could convert 100 g/L lactose into 46.1 ± 1.4 g/L ethanol in DWP, a yield of 88% of the theoretical. The resting cells could be re-used at least three times, and the ethanol productivities obtained were 0.96 g/L/h, 2.2 g/L/h, and 1.6 g/L/h, respectively., Conclusions: An efficient process for producing ethanol from DWP, based on C. glutamicum, was demonstrated. The results obtained clearly show a great potential for this newly developed platform for producing value-added chemicals from dairy waste.

Background: Early antibiotic switch and early discharge protocols have not been widely studied in Latin America. Our objective was to describe real-world treatment patterns, resource use, and estimate opportunities for early switch from intravenous to oral antibiotics and early discharge for patients hospitalized with methicillin-resistant Staphylococcus aureus complicated skin and soft-tissue infections., Materials/methods: This retrospective medical chart review recruited 72 physicians from Brazil to collect data from patients hospitalized with documented methicillin-resistant Staphylococcus aureus complicated skin and soft tissue infections between May 2013 and May 2015, and discharged alive by June 2015. Data collected included clinical characteristics and outcomes, hospital length of stay, methicillin-resistant Staphylococcus aureus-targeted intravenous and oral antibiotic use, and early switch and early discharge eligibility using literature-based and expert-validated criteria., Results: A total of 199 patient charts were reviewed, of which 196 (98.5%) were prescribed methicillin-resistant Staphylococcus aureus -active therapy. Only four patients were switched from intravenous to oral antibiotics while hospitalized. The mean length of methicillin-resistant Staphylococcus aureus-active treatment was 14.7 (standard deviation, 10.1) days, with 14.6 (standard deviation, 10.1) total days of intravenous therapy. The mean length of hospital stay was 22.2 (standard deviation, 23.0) days. The most frequent initial methicillin-resistant Staphylococcus aureus-active therapies were intravenous vancomycin (58.2%), intravenous clindamycin (19.9%), and intravenous daptomycin (6.6%). Thirty-one patients (15.6%) were discharged with methicillin-resistant Staphylococcus aureus -active antibiotics of which 80.6% received oral antibiotics. Sixty-two patients (31.2%) met early switch criteria and potentially could have discontinued intravenous therapy 6.8 (standard deviation, 7.8) days sooner, and 65 patients (32.7%) met early discharge criteria and potentially could have been discharged 5.3 (standard deviation, 7.0) days sooner., Conclusions: Only 2% of patients were switched from intravenous to oral antibiotics in our study while almost one-third were early switch eligible. Additionally, one-third of hospitalized patients with methicillin-resistant Staphylococcus aureus complicated skin and soft tissue infections were early discharge eligible indicating opportunity for reducing intravenous therapy and days of hospital stay. These results provide insight into possible benefits of implementation of early switch/early discharge protocols in Brazil., (Copyright © 2019 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Strain development is frequently used to improve the performance and functionality of industrially important microbes. As traditional mutagenesis screen is especially utilized by the food industry to improve strains used in food fermentation, high-throughput and cost-effective screening tools are important in mutant selection. The emerging droplet-based microfluidics technology miniaturizes the volume for cell cultivation and phenotype interrogation down to the picoliter scales, which facilitates screening of microbes for improved phenotypical properties tremendously. In this mini review, we present recent application of the droplet-based microfluidics in microbial strain improvement with a focus on its potential use in the screening of lactic acid bacteria.

Background: In this analysis we compared quality-adjusted survival outcomes between nab-paclitaxel (nab-P) and standard paclitaxel (Pac) using data from the nab-P phase III registration trial in metastatic breast cancer., Patients and Methods: Quality-adjusted overall survival was estimated using the quality-adjusted time without symptoms or toxicity (Q-TWiST) approach. Overall survival was partitioned into time without progression/Grade ≥ 3 adverse events (AEs) toxicity (TWiST), time with Grade ≥ 3 AE toxicity (TOX), and time after relapse (REL). Q-TWiST was calculated by multiplying mean time in each health state by its assigned utility (base-case utility values: time without symptoms of disease progression or toxicity of Grade ≥ 3 adverse events [TWiST] = 1.0, TOX = 0.5, and REL = 0.5). In threshold analyses, TOX and REL varied from 0.0 to 1.0 whereas TWiST was maintained at 1.0. Comparisons were made for the intent-to-treat population and the subset of patients initiating the study drugs as second or subsequent lines (2L+) of chemotherapy (per approved nab-P indication; 2L+ subpopulation). A ≥ 15% relative Q-TWiST gain (vs. mean Pac overall survival) was considered clearly clinically important., Results: In the intent-to-treat population, nab-P (n = 229) versus Pac (n = 225) resulted in nonsignificant gains of 1.4 months of mean Q-TWiST (11.6 vs. 10.2 months; 95% confidence interval [CI], -0.03 to 2.8). In the 2L+ subpopulation, nab-P (n = 132) versus Pac (n = 136) resulted in a statistically significant gain of 2.2 months of mean Q-TWiST (10.5 vs. 8.4 months; 95% CI, 0.6-3.8), with a 17.1% relative Q-TWiST gain (threshold analysis range, 14.0%-19.5%, both figures significant)., Conclusion: In its approved indication for metastatic breast cancer, nab-P showed a statistically significant and clearly clinically important improvement in quality-adjusted survival time versus Pac in the 2L+ subpopulation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Evolution, i.e. the change in heritable characteristics of biological populations over successive generations, has created the diversity of life that exists today. In this study we have harnessed evolution to create faster growing mutants of Corynebacterium glutamicum, i.e. to debottleneck growth rate of this highly important industrial workhorse. After approximately 1500 generations of Adaptive Laboratory Evolution (ALE) in defined minimal medium with glucose, we obtained faster growing mutants with specific growth rate as high as 0.64 h -1 as compared with 0.45 h -1 for the wild type, and this 42% improvement is the highest reported for C. glutamicum to date. By genome resequencing and inverse metabolic engineering, we were able to pinpoint two mutations contributing to most of the growth improvement, and these resided in the transcriptional regulators GntR1 (gntR1-E70K) and RamA (ramA-A52V). We confirmed that the two mutations lead to alteration rather than elimination of function, and their introduction in the wild-type background resulted in a specific growth rate of 0.62 h -1 . The glycolytic and pentose phosphate pathway fluxes had both increased significantly, and a transcriptomic analyses supported this to be associated with increased capacity. Interestingly, the observed fast growth phenotype was not restricted to glucose but was also observed on fructose, sucrose and xylose, however, the effect of the mutations could only be seen in minimal medium, and not rich BHI medium, where growth was already fast. We also found that the mutations could improve the performance of resting cells, under oxygen-deprived conditions, where an increase in sugar consumption rate of around 30% could be achieved. In conclusion, we have demonstrated that it is feasible to reprogram C. glutamicum into growing faster and thus enhance its industrial potential., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD + , and a high titer of 371mM (32g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361mM (32g/L) R-BDO with a yield of 81% or 365mM (33g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. We have developed a tool for this purpose for the important industrial workhorse Corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. We first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pJS31 serving as the carrier. The pJS31 vector contains a synthetic cassette including a phage attachment site attP for integration, a bacterial attachment site attB for subsequent integration, a multiple cloning site, and two modified loxP sites to facilitate easy removal of undesirable vector elements. Meanwhile, we constructed a derivative of the wild-type strain ATCC 13032 carrying an attB site in its chromosome (JS34) and demonstrated that pJS31 readily could integrate into the attB site in this strain providing expression of the corresponding integrase. Subsequent expression of the Cre recombinase promoted recombination between the modified loxP sites, resulting in a strain only retaining the target insertions and an attB site. To simplify the procedure, non-replicating circular expression units for the phage integrase and the Cre recombinase were used. As a showcase, we used the tool to construct a battery of strains simultaneously expressing the two reporter genes, lacZ (encoding β-galactosidase) and gusA (encoding β-glucuronidase), to arbitrary levels. In principle, an unlimited number of genes, whether native, heterologous, or synthetic, can be introduced using the developed approach, and this should greatly facilitate metabolic optimization of this important platform organism.

Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B 2 (riboflavin). Initially, we used a roseoflavin-resistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes. IMPORTANCE The food industry prefers to use classical approaches, e.g., random mutagenesis followed by screening, to improve microorganisms used in food production, as the use of recombinant DNA technologies is still not widely accepted. Although modern automated screening platforms are widely accessible, screening remains as a bottleneck in strain development, especially when a mild mutagenesis approach is applied to reduce the chance of accumulating unintended mutations, which may cause unwanted phenotypic changes. Here, we incorporate a droplet-based high-throughput screening method into the strain development process and readily capture L. lactis variants with more efficient vitamin secretion from low-error-rate mutagenesis libraries. This study shows that useful mutants showing strong phenotypes but without extensive mutations can be identified with efficient screening technologies. It is therefore possible to avoid accumulating detrimental mutations while enriching beneficial ones through iterative mutagenesis screening. Due to the low mutation rates, the genetic determinants are also readily identified., (Copyright © 2017 Chen et al.)

Biocompatible chemistry (BC), that is, non-enzymatic chemical reactions compatible with living organisms, is increasingly used in conjunction with metabolically engineered microorganisms for producing compounds that do not usually occur naturally. Here we report production of one such compound, (3S)-acetoin, a valuable precursor for chiral synthesis, using a metabolically engineered Lactococcus lactis strain growing under respiratory conditions with ferric iron serving as a BC component. The strain used has all competing product pathways inactivated, and an appropriate cofactor balance is achieved by fine-tuning the respiratory capacity indirectly via the hemin concentration. We achieve high-level (3S)-acetoin production with a final titer of 66 mM (5.8 g/L) and a high yield (71% of the theoretical maximum). To the best of our knowledge, this is the first report describing production of (3S)-acetoin from sugar by microbial fermentation, and the results obtained confirm the potential that lies with BC for producing useful chemicals. Biotechnol. Bioeng. 2016;113: 2744-2748. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The potential that lies in harnessing the chemical synthesis capabilities inherent in living organisms is immense. Here we demonstrate how the biosynthetic machinery of Lactococcus lactis, can be diverted to make (3R)-acetoin and the derived 2,3-butanediol isomers meso-(2,3)-butanediol (m-BDO) and (2R,3R)-butanediol (R-BDO). Efficient production of (3R)-acetoin was accomplished using a strain where the competing lactate, acetate and ethanol forming pathways had been blocked. By introducing different alcohol dehydrogenases into this strain, either EcBDH from Enterobacter cloacae or SadB from Achromobacter xylosooxidans, it was possible to achieve high-yield production of m-BDO or R-BDO respectively. To achieve biosustainable production of these chemicals from dairy waste, we transformed the above strains with the lactose plasmid pLP712. This enabled efficient production of (3R)-acetoin, m-BDO and R-BDO from processed whey waste, with titers of 27, 51, and 32 g/L respectively. The corresponding yields obtained were 0.42, 0.47 and 0.40 g/g lactose, which is 82%, 89%, and 76% of maximum theoretical yield respectively. These results clearly demonstrate that L. lactis is an excellent choice as a cell factory for transforming lactose containing dairy waste into value added chemicals.

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F 1 -ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F 1 -ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F 1 -ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F 1 -ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F 1 -ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.

Objectives: Adopting a unique Spanish perspective, this study aims to assess healthcare resource utilization (HCRU) and the costs of treating nosocomial pneumonia (NP) produced by methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized adults using linezolid or vancomycin. An evaluation is also made of the renal failure rate and related economic outcomes between study groups., Design: An economic post hoc evaluation of a randomized, double-blind, multicenter phase 4 study was carried out., Scope: Nosocomial pneumonia due to MRSA in hospitalized adults., Participants: The modified intent to treat (mITT) population comprised 224 linezolid- and 224 vancomycin-treated patients., Interventions: Costs and HCRU were evaluated between patients administered either linezolid or vancomycin, and between patients who developed renal failure and those who did not., Primary Endpoints: Analysis of HCRU outcomes and costs., Results: Total costs were similar between the linezolid- (€17,782±€9,615) and vancomycin-treated patients (€17,423±€9,460) (P=.69). The renal failure rate was significantly lower in the linezolid-treated patients (4% vs. 15%; P<.001). The total costs tended to be higher in patients who developed renal failure (€19,626±€10,840 vs. €17,388±€9,369; P=.14). Among the patients who developed renal failure, HCRU (days on mechanical ventilation: 13.2±10.7 vs. 7.6±3.6 days; P=.21; ICU stay: 14.4±10.5 vs. 9.9±6.6 days; P=.30; hospital stay: 19.5±9.5 vs. 16.1±11.0 days; P=.26) and cost (€17,219±€8,792 vs. €20,263±€11,350; P=.51) tended to be lower in the linezolid- vs. vancomycin-treated patients. There were no statistically significant differences in costs per patient-day between cohorts after correcting for mortality (€1000 vs. €1,010; P=.98)., Conclusions: From a Spanish perspective, there were no statistically significant differences in total costs between the linezolid and vancomycin pneumonia cohorts. The drug cost corresponding to linezolid was partially offset by fewer renal failure adverse events., (Copyright © 2016 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.)

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95mM or 8.2g/L) and high yield (87% of the theoretical maximum). Subsequently, the pathway was extended to (S,S)-2,3-butanediol (S-BDO) through efficiently linking two metabolic pathways via chemical catalysis. This resulted in efficient homo-S-BDO production with a titer of 74mM (6.7g/L) S-BDO and a yield of 82%. The diacetyl and S-BDO production rates and yields obtained are the highest ever reported, demonstrating the promising combination of metabolic engineering and biocompatible chemistry as well as the great potential of L. lactis as a new production platform., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Sustainable and economically feasible ways to produce ethanol or other liquid fuels are becoming increasingly relevant due to the limited supply of fossil fuels and the environmental consequences associated with their consumption. Microbial production of fuel compounds has gained a lot of attention and focus has mostly been on developing bio-processes involving non-food plant biomass feedstocks. The high cost of the enzymes needed to degrade such feedstocks into its constituent sugars as well as problems due to various inhibitors generated in pretreatment are two challenges that have to be addressed if cost-effective processes are to be established. Various industries, especially within the food sector, often have waste streams rich in carbohydrates and/or other nutrients, and these could serve as alternative feedstocks for such bio-processes. The dairy industry is a good example, where large amounts of cheese whey or various processed forms thereof are generated. Because of their nutrient-rich nature, these substrates are particularly well suited as feedstocks for microbial production., Results: We have generated a Lactococcus lactis strain which produces ethanol as its sole fermentation product from the lactose contained in residual whey permeate (RWP), by introducing lactose catabolism into a L. lactis strain CS4435 (MG1363 Δ(3) ldh, Δpta, ΔadhE, pCS4268), where the carbon flow has been directed toward ethanol instead of lactate. To achieve growth and ethanol production on RWP, we added corn steep liquor hydrolysate (CSLH) as the nitrogen source. The outcome was efficient ethanol production with a titer of 41 g/L and a yield of 70 % of the theoretical maximum using a fed-batch strategy. The combination of a low-cost medium from industrial waste streams and an efficient cell factory should make the developed process industrially interesting., Conclusions: A process for the production of ethanol using L. lactis and a cheap renewable feedstock was developed. The results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) and corn milling industry (CSLH) to ethanol and the process developed shows great potential for commercial realization.

This retrospective medical chart review aimed to provide a current, real-world overview of biologic usage in patients with rheumatoid arthritis (RA) in Germany, Spain, and the UK, and estimate clinical and healthcare utilization outcomes associated with early versus late treatment. Adults (≥18 years) with a confirmed RA diagnosis between January 2008 and December 2010, who received biologic treatment for ≥3 months and had ≥12 months of follow-up were included. Early treatment was receipt of biologic agent ≤1 year after RA diagnosis. Outcomes included 28-joint disease activity score (DAS28) reduction of ≥1.2 from biologic start and remission (DAS28 < 2.6). Time to outcome was evaluated using Kaplan-Meier curves and log-rank tests. Of 328 patients enrolled (Germany [n = 111], Spain [n = 106], UK [n = 111]), 58.2 % received early biologic (Germany: 55.0 %, UK: 55.9 %, Spain: 64.2 %; p = 0.321). First-line biologics were more frequent in Spain (26.4 %) and Germany (19.8 %) versus the UK (7.2 %; p < 0.001). Late-treated patients were hospitalized more often than early-treated patients (10.5 vs 2.9 % [p = 0.006] for 9.0 vs 5.4 mean inpatient days [p = 0.408]). DAS28 was 5.1 at biologic initiation (n = 310); 73.5 % of patients had a DAS28 decrease of ≥1.2 and 44.5 % achieved remission. More patients had DAS28 decrease of ≥1.2 (79.2 vs 65.9 %; p = 0.009) and remission (51.1 vs 35.6 %; p = 0.007) with early versus late treatment, with a significant difference in Kaplan-Meier curves when indexing on time since diagnosis (p < 0.001) and biologic start (p = 0.024). In RA patients receiving biologic therapy, over half received biologic therapy early. Early initiation was associated with improved clinical outcomes and reduced hospitalization rates versus late treatment.

Unlabelled: The trxB2 gene, which is annotated as a thioredoxin reductase, was found to be essential for growth of Lactococcus lactis in the presence of oxygen. The corresponding protein (TrxB2) showed a high similarity with Bacillus subtilis YumC (E value = 4.0E-88), and YumC was able to fully complement the ΔtrxB2 mutant phenotype. YumC represents a novel type of ferredoxin (flavodoxin) reductase (FdR) with hitherto-unknown biological function. We adaptively evolved the ΔtrxB2 mutant under aerobic conditions to find suppressor mutations that could help elucidate the involvement of TrxB2 in aerobic growth. Genome sequencing of two independent isolates, which were able to grow as well as the wild-type strain under aerated conditions, revealed the importance of mutations in nrdI, encoding a flavodoxin involved in aerobic ribonucleotide reduction. We suggest a role for TrxB2 in nucleotide metabolism, where the flavodoxin (NrdI) serves as its redox partner, and we support this hypothesis by showing the beneficial effect of deoxynucleosides on aerobic growth of the ΔtrxB2 mutant. Finally, we demonstrate, by heterologous expression, that the TrxB2 protein functionally can substitute for YumC in B. subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant., Importance: Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently identified bacterial YumC-type FdR belongs to a novel type, the biological function of which still remains elusive. We found that the YumC-like FdR (TrxB2) is essential for aerobic growth of Lactococcus lactis. We suggest that the YumC-type FdR is involved in the ribonucleotide reduction by the class Ib ribonucleotide reductase, which represents the workhorse for the bioconversion of nucleotides to deoxynucleotides in many prokaryotes and eukaryotic pathogens under aerobic conditions. As the partner of the flavodoxin (NrdI), the key FdR is missing in the current model describing the class Ib system in Escherichia coli. With this study, we have established a role for this novel type of FdR and in addition found the missing link needed to explain how ribonucleotide reduction is carried out under aerobic conditions., (Copyright © 2015 Chen et al.)